Methods and materials for generating CD8+ T cells having the ability to recognize cancer cells expressing a HER2/neu polypeptide

ABSTRACT

This document provides methods and materials for generating CD8 −  T cells having the ability to recognize cancer cells expressing a HER2/neu polypeptide. For example, methods and materials for using a polypeptide consisting of an SLAFLPESFD amino acid sequence in vivo or in vitro to generate CD8 +  T cells having the ability to recognize and lyse cancer cells expressing a HER2/neu polypeptide are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/379,150, filed Aug. 15, 2014, which is a National Stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2013/026484, having an International Filing Date of Feb. 15, 2013,which claims the benefit of U.S. Provisional Application Ser. No.61/600,480, filed Feb. 17, 2012. The disclosures of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA113861 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND 1. Technical Field

This document relates to methods and materials for generating CD8⁺ Tcells having the ability to recognize cancer cells expressing a HER2/neupolypeptide. For example, this document relates to methods and materialsfor using a polypeptide consisting of an SLAFLPESFD amino acid sequencein vivo or in vitro to generate CD8⁺ T cells having the ability torecognize cancer cells expressing a HER2/neu polypeptide.

2. Background Information

Cancer vaccines have the ability to stimulate or restore the immunesystem so that it can fight cancer. In some cases, cancer vaccines canbe designed to treat an existing cancer by strengthening the patient'sdefenses against the cancer.

SUMMARY

This document provides methods and materials for generating CD8⁺ T cellshaving the ability to recognize cancer cells expressing a HER2/neupolypeptide. For example, this document provides methods and materialsfor using a polypeptide consisting of an SLAFLPESFD amino acid sequence(SEQ ID NO:1) in vivo or in vitro to generate CD8⁺ T cells having theability to recognize and lyse cancer cells expressing a HER2/neupolypeptide. A polypeptide consisting of an SLAFLPESFD (SEQ ID NO:1)amino acid sequence can be referred to as the SLAFLPESFD (SEQ ID NO:1)polypeptide, the p373-382 polypeptide, or a polypeptide consisting ofthe amino acid sequence set forth in SEQ ID NO:1. As described herein,the SLAFLPESFD (SEQ ID NO:1) polypeptide or a vaccine compositioncontaining the SLAFLPESFD (SEQ ID NO:1) polypeptide can be administeredto a cancer patient having cancer cells that express a HER2/neupolypeptide under conditions wherein the patient produces CD8⁺ T cellshaving the ability to recognize and lyse those cancer cells. In somecases, such CD8⁺ T cells can be referred to as CD8⁺ T cells generatedusing the SLAFLPESFD (SEQ ID NO:1) polypeptide.

Having the ability to generate CD8⁺ T cells with the ability torecognize and lyse cancer cells that express a HER2/neu polypeptide canallow clinicians to provide cancer patients with additional effectivetreatment options. For example, the vaccines provided herein can be usedalone or in combination with other cancer treatment options to providecancer patients with an effective population of CD8⁺ T cells designed tokill cancer cells that express a HER2/neu polypeptide.

In general, one aspect of this document features a polypeptide (e.g., apurified polypeptide), wherein the sequence of the polypeptide consistsof the amino acid sequence set forth in SEQ ID NO:1. In some case, thepolypeptide can include an N and/or C terminal modification.

In another aspect, this document features a vaccine compositioncomprising, or consisting essentially of, a polypeptide (e.g., apurified polypeptide), wherein the sequence of the polypeptide consistsof the amino acid sequence set forth in SEQ ID NO:1. In some case, thepolypeptide can include an N and/or C terminal modification. Thecomposition can comprise an adjuvant. The adjuvant can be an oil andwater mixture. The adjuvant can be Montanide ISA-51. The composition cancomprise IL-2, IL-12, GM-CSF, or rintatolimod.

In another aspect, this document features a method for increasing thenumber of CD8+ T cells having the ability to kill cancer cellsexpressing a HER2/neu polypeptide. The method comprises, or consistsessentially of, contacting a population of CD8⁺ T cells with apolypeptide (e.g., a purified polypeptide), wherein the sequence of thepolypeptide consists of the amino acid sequence set forth in SEQ IDNO:1. The contacting step can occur in an ex vivo manner. The contactingstep can occur in an in vivo manner.

In another aspect, this document features a method for increasing,within a human, the number of CD8⁺ T cells having the ability to killcancer cells expressing a HER2/neu polypeptide. The method comprises, orconsists essentially of, administering a vaccine composition to thehuman, wherein the composition comprises, or consists essentially of, apolypeptide (e.g., a purified polypeptide), wherein the sequence of thepolypeptide consists of the amino acid sequence set forth in SEQ IDNO:1. In some case, the polypeptide can include an N and/or C terminalmodification. The human can contain cancer cells expressing the HER2/neupolypeptide. The composition can comprise an adjuvant. The adjuvant canbe an oil and water mixture. The adjuvant can be Montanide ISA-51. Thecomposition can comprise IL-2, IL-12, GM-CSF, or rintatolimod. Themethod can comprise administering IL-2, IL-12, GM-CSF, rintatolimod, ora combination thereof to the human. The method can further compriseadministering trastuzumab to the human.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A shows example total ion chromatograms identifying polypeptidesderived from HER-2/neu 19-mer polypeptide (FAGCKKIFGSLAFLPESFD) (SEQ IDNO:3) by proteasomal or immunoproteasomal catalysis.

FIG. 1B shows example extracted ion chromatograms from the reactionsdescribed in FIG. 1A examining processing of HER-2/neu p369-377 from the19-mer polypeptide.

FIG. 1C shows example extracted ion chromatograms demonstrating thatp373-382 (SLAFLPESFD) (SEQ ID NO:1) is processed from aHER-2/neu-derived 23-mer (QEFAGCKKIFGSLAFLPESFDGD; SEQ ID NO:19) byproteasomal or immunoproteasomal catalysis.

FIG. 2A shows HER-2/neu polypeptide p373-382 binding to TAP deficient T2cells.

FIG. 2B shows dose dependent saturable binding of HER-2/neu polypeptidep373-382 to T2 cells.

FIG. 2C shows relative binding affinities of HER-2/neu polypeptidep373-382 and p369-377 compared to high affinity influenza-derived HLA-A2binding polypeptide, GILGFVFTL (p58-66; SEQ ID NO:20).

FIG. 3A shows IFN-γ ELIspot analysis showing that CD8 T cells generatedagainst HER-2/neu polypeptide p373-382 respond to autologous cellsloaded with either p373-382 or p369-377.

FIG. 3B shows IFN-γ ELIspot analysis demonstrating that CD8 T cellsgenerated against HER-2/neu polypeptide p373-382 respond toHER-2/neu-expressing tumor cells with release of IFN-γ.

FIG. 3C shows cytotoxic T cell analysis demonstrating that CD8 T cellsgenerated against HER-2/neu polypeptide p373-382 lyseHER-2/neu-expressing tumor cells.

FIGS. 4A-B shows, using IFN-γ ELIspot analysis, that the IFN-γ responseof CD8 T cells generated against HER-2/neu polypeptide p373-382 isblocked by MEW class I blocking antibodies (FIG. 4A: anti-HLA-A2; andFIG. 4B: anti-HLA-ABC).

FIGS. 4C-D shows, using cytotoxic T cell assays, that the lysis responseof CD8 T cells generated against HER-2/neu polypeptide p373-382 isblocked by MEW class I blocking antibodies (FIG. 4C: anti-HLA-A2; andFIG. 4D: anti-HLA-ABC).

DETAILED DESCRIPTION

This document provides methods and materials for generating CD8⁺ T cellshaving the ability to recognize cancer cells expressing a HER2/neupolypeptide. For example, this document provides methods and materialsfor using a polypeptide consisting of the amino acid sequence set forthin SEQ ID NO:1 in vivo or in vitro to generate CD8⁺ T cells having theability to recognize and lyse cancer cells expressing a HER2/neupolypeptide. In some cases, this document provides the SLAFLPESFD (SEQID NO:1) polypeptide and vaccine compositions containing the SLAFLPESFD(SEQ ID NO:1) polypeptide as well as methods for using the SLAFLPESFDpolypeptide or vaccine compositions containing the SLAFLPESFD (SEQ IDNO:1) polypeptide to generate CD8⁺ T cells having the ability torecognize cancer cells expressing a HER2/neu polypeptide.

In some cases, a polypeptide provided herein (e.g., a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO:1) can beused in combination with dendritic cells to treat cancer. For example,dendritic cells contacted with the SLAFLPESFD (SEQ ID NO:1) polypeptidecan be used to treat cancer.

The SLAFLPESFD (SEQ ID NO:1) polypeptide provided herein can besubstantially pure. The term “substantially pure” with respect to apolypeptide refers to a polypeptide that has been separated fromcellular components with which it is naturally accompanied. For example,a synthetically generated polypeptide can be a substantially purepolypeptide. Typically, a polypeptide provided herein is substantiallypure when it is at least 60 percent (e.g., 65, 70, 75, 80, 90, 95, or 99percent), by weight, free from proteins and naturally-occurring organicmolecules with which it is naturally associated. In general, asubstantially pure polypeptide will yield a single major band on anon-reducing polyacrylamide gel.

The SLAFLPESFD (SEQ ID NO:1) polypeptide provided herein can be preparedin a wide variety of ways. Because of its relatively short size, theSLAFLPESFD (SEQ ID NO:1) polypeptide can be synthesized in solution oron a solid automatic synthesizer in accordance with known protocols.See, for example, Stewart and Young, Solid Phase Polypeptide Synthesis,2d. ed., Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc.,105:6442 (1983); Merrifield, The Polypeptides, Gross and Meienhofer,ed., academic Press, New York, pp. 1-284 (1979). In some cases, apolypeptide provided herein (e.g., a SLAFLPESFD (SEQ ID NO:1)polypeptide) can be synthesized with either an amide (e.g., NH₂) or freeacid (e.g., COOH) C terminus, both of which can have the ability to bindHLA-A2.

In some cases, recombinant DNA technology can be used wherein a nucleicacid sequence that encodes a SLAFLPESFD (SEQ ID NO:1) polypeptideprovided herein is inserted into an expression vector, introduced (e.g.,by transformation or transfection) into an appropriate host cell, andcultivated under conditions suitable for expression. These proceduresare generally known in the art, as described generally in Sambrook etal., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1982), and Ausubel et al., (ed.) CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc., New York(1987), and U.S. Pat. Nos. 4,237,224; 4,273,875; 4,431,739; 4,363,877;and 4,428,941, for example.

This document also provides polypeptides (e.g., substantially purepolypeptides) consisting of one of the amino acid sequences set forth inTable 1. Such polypeptides can be made and used in the same mannerdescribed herein for the SLAFLPESFD (SEQ ID NO:1) polypeptide.

In some cases, the polypeptides provided herein can be incubated with apopulation of CD8⁺ T cells to generate an activated pool of CD8⁺ T cellsthat have the ability to recognize p373-382 or a HER2/neu polypeptide.For example, one or more of the polypeptides provided herein (e.g., theSLAFLPESFD (SEQ ID NO:1) polypeptide) can be used in an ex vivo mannerto created antigen-specific CD8⁺ T cells that can be used to treatcancers. In some cases, the polypeptides provided herein can be used togenerate a pool of activated HER2/neu polypeptide-specific CD8⁺ T cellsthat can be used alone, or in combination with monoclonal antibodytherapy, CTL therapy, or both monoclonal antibody therapy and CTLtherapy, to treat cancer. For example, an anti-HER-2/neu monoclonalantibody therapy can be combined with infusion of CD8⁺ T cells generatedusing p373-382 polypeptides to treat cancer (e.g., breast cancer). Insome cases, a Herceptin (trastuzumab) therapy can be combined withinfusion of CD8⁺ T cells generated using p373-382 polypeptides to treatcancer (e.g., breast cancer).

This document also provides vaccine compositions that contain animmunogenically effective amount of one or more of the polypeptidesprovided herein. A vaccine composition provided herein can be used bothas a preventative or therapeutic vaccine. The vaccine compositionsprovided herein can be administered and formulated using any appropriatetechniques including, without limitation, those techniques describedelsewhere (see, e.g., [0132]-[0173] of U.S. Patent ApplicationPublication No. 2010-0310640).

In some cases, a vaccine composition provided herein can include GM-CSF(e.g., sargramostim), rintatolimod (e.g., Ampligen®), IL-2, IL-12, anadjuvant, or a combination thereof. For example, a vaccine compositionprovided herein can include GM-CSF and an adjuvant. Examples ofadjuvants include, without limitation, CpG oligonucleotides,monophosphoryl lipid A, and Montanide ISA-51. In some cases, theadjuvant can be an oil and water mixture such as Montanide ISA-51.

In some cases, a vaccine composition provided herein can include acombination of polypeptides. For example, a vaccine composition providedherein can include the SLAFLPESFD polypeptide and/or one or more of theother polypeptides set forth in Table 1 in combination with one or morepolypeptides set forth in U.S. Patent Application Publication No.2010-0310640, the Karyampudi et al. reference (Clin. Cancer Res.,16(3):825-34 (2010)), the Holmes et al. reference (J. Clin. Oncol.,26(20):3426-33 (2008)), the Gritzapis et al. reference (Vaccine,28(1):162-70 (2009)), the Perez et al. reference (Cancer Immunol.Immunother., 50(11):615-24 (2002)), the Knutson et al. reference (J.Clin. Invest., 107(4):477-84 (2001)), or the Salazar et al. reference(Clin. Cancer Res., 9(15):5559-65 (2003)).

Any appropriate method can be used to administer a vaccine compositionprovided herein to a mammal (e.g., a human). For example, a vaccinecomposition or polypeptide provided herein can be administered alone orin combination with other polypeptides in doses ranging from 100 to10,000 micrograms given by intradermal or subcutaneous routes monthlyfor a total of four to twelve months (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or12 months).

The methods and materials provided herein can be used to treat any typeof cancer that expresses a HER2/neu polypeptide. For example, themethods and materials provided herein can be used to treat breastcancer, ovarian cancer, colon cancer, esophageal cancer, or lung cancer.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Identification of a Potent Class I MHC MoleculeEpitope of a HER2/neu Polypeptide

HER-2/neu is a polypeptide that is overexpressed in a wide variety ofcancers and is a therapeutic target, particularly in breast cancer. Forexample, NeuVax is a previously developed vaccine that includes theHER-2/neu-derived polypeptide p369-377 (amino acid sequence: KIFGSLAFL(SEQ ID NO:2), also called E75) derived from the extracellular domain ofHER-2/neu and mixed with GM-CSF. This vaccine aims to prime the immuneresponse against E75 so that T cells are generated that can recognizeand kill the patient's cancer cells, which are presumably displaying thesame peptides on their cell surface in association with MHC class Imolecules.

The following was performed to determine if E75 is processed fromHER-2/neu or HER-2/neu fragments by multi-subunit enzymes referred to asthe proteasome and immunoproteasome, which are required for loading ofpolypeptide onto MHC class I molecules. To determine this, the 19-merpolypeptide, FAGCKKIFGSLAFLPESFD (SEQ ID NO:3), was synthesized. Thispolypeptide matches HER-2/neu amino acids 364-382, and it fully containsE75 (underlined). The 19-mer was then cleaved using purified 20Sproteasome and immunoproteasome. Although E75 is reported to be cleavedas determined by proteasome algorithms, the in vitro data presentedherein did not reveal processing of this polypeptide from longerHER-2/neu polypeptides (FIGS. 1A and 1B). However, the 19-merpolypeptide was consistently found to be processed into several othershorter polypeptides (FIG. 1A and Table 1). None of these shorterpolypeptides scored as highly as E75 for binding to HLA-A2 using themathematical algorithms (Table 1).

TABLE 1 HER-2/neu Peptide Peptide (SYFPEITHI) Cleavage amino retentiongenerated generated HLA-A*0201 predicted FIG. 1A acid time by immuno- bybinding by ip/p label number (min) Peptide proteasome proteasome scoresewers 1 371-381 20.4  FGSLAFLPESF + + NA Y (SEQ ID NO: 4) 2 364-37415.45 FAGCKKIFGSL + + NA Y (SEQ ID NO: 5) 3 372-382 18.4 GSLAFLPESFD + + NA Y (SEQ ID NO: 6) 4 373-382 18.5  SLAFLPESFD + + 13 N(SEQ ID NO: 1) 5 371-380 19.1  FGSLAFLPES + +  8 N (SEQ ID NO: 7) 5372-381 19.1  GSLAFLPESF + +  5 Y (SEQ ID NO: 8) 6 364-373 12.8 FAGCKKIFGS + + 10 N (SEQ ID NO: 9) 7 374-382 18.2  LAFLPESFD + +  8 N(SEQ ID NO: 10) 8 373-381 19.2  SLAFLPESF + + 16 N (SEQ ID NO: 11)369-377 NA KIFGSLAFL - - 28 Y (SEQ ID NO: 2) 9 375-382 16.06AFLPESFD + + NA Y (SEQ ID NO: 12) * 364-382 19.14 FAGCKKIFGSLAFLP NA NANA NA ESFD-NH2 (SEQ ID NO: 13) * 364-382 19.14 FAGCKKIFGSLAFLP NA NA NANA ESFD-COOH (SEQ ID NO: 14) ** NA 19.54 FAGKKIFGSLAFLPE NA NA NA NASFD-NH2 (SEQ ID NO: 15) ** NA 19.54 FAGKKIFGSLAFLPE NA NA NA NA SFD-COOH(SEQ ID NO: 16) *** NA 14.78 FAGKKIFGSL NA NA NA NA (SEQ ID NO: 17) ***NA 14.78 GKKIFGSLAF NA NA NA NA (SEQ ID NO: 18) A 19 mer sequence fromHER-2/neu is processed into smaller polypeptide fragments by theimmunoproteasome and proteasome, and these fragments are predicted tobind HLA-A*0201. The symbol (+) indicates the polypeptide was producedby the respective enzyme in an in vitro assay. The symbol (-) indicateslack of peptide detection in an in vitro assay in samples containing therespective enzyme. The SYFPEITHI server was used to predict nonamer anddecamer polypeptide binding to HLA-A*0201. The 20S and C-term 3.0prediction methods on the Netchop 3.1 server and the Proteasome CleavagePrediction Server with models 1, 2, and 3 for the proteasome andimmunoproteasome enzymes were used to predict whether the smallerpolypeptide could be processed by the enzymes from the larger 19 mersequence, irrespective of in vitro data. NA, not applicable, indicatesthat the polypeptide is either a deletion product, is starting materialand thus would not be generated in the assay, or is too large forbinding predictions to HLA-A*0201. Numbers and asterisks indicatepeptide labels in FIG. 1A.

One of the observed processed polypeptides (p373-382) represented theend terminal ten amino acids of the 19-mer. To determine if thispolypeptide could be processed from larger peptides, a 23-mer containingp373-382 was synthesized and treated with the proteasomes. As shown inFIG. 1C, p373-382 was indeed released from the 23-mer.

Many of the polypeptides that were processed in the assay weresynthesized and tested for binding to the class I MHC molecule, HLA-A2,using the standard T2 HLA-A2 stabilization assay. HLA-A2 is an MHC classI molecule that is prevalent in approximately 30-40% of the Caucasianpopulation. It is often used as a target in vaccine trials since it hasthe potential to benefit a large number of breast cancer patients. Oneof the synthesized polypeptides, p373-382 (SLAFLPESFD), was able tostrongly bind the HLA-A2 molecule, at levels comparable to the positivecontrol, a polypeptide from the influenza virus (FLU) (FIG. 2).Surprisingly, even low levels of p373-382 bound, compared to p369-377,which required higher concentrations (FIG. 2).

Taken together, these results demonstrate that the HER-2/neu polypeptidep373-382 is processed from longer HER-2/neu polypeptides and bindsHLA-A2.

An ELIspot was performed to determine if the p373-382 epitope isnaturally processed by the cellular machinery in cancer cells and todetermine if it has the potential of being bound to HLA-A2 on thesurface of cancer cells, where it can serve as a target for primed andactivated immune cells. If specific immune cells can recognize thep373-382:HLA-class I complex on cancer cells, then they can kill thecancer cells and prevent the cancer from progressing in patients. Sinceit was shown that the p373-382 polypeptide was processed in vitro bycellular machinery and was capable of binding strongly to HLA-A2molecules, an ELIspot was performed to determine whether CD8⁺ T cellsgenerated using the p373-382 polypeptide can be generated and if these Tcells are able to recognize HER-2/neu⁻ breast cancer cells.

FIG. 3A reveals that that CD8⁺ T cell lines were generated using thepFLU polypeptide (control), p369-377, and p373-382. The control FLU Tcells only recognized target cells pulsed with FLU polypeptide, asexpected. HER-2/neu p369-377 polypeptide-generated T cells recognizedtarget cells pulsed with p369-377 polypeptide and target cells pulsedwith p373-382. p373-382-generated T cells recognized target cells pulsedwith either the p373-382 polypeptide or the p369-377 polypeptide,indicating that there is cross-reactivity between the two polypeptideswhich could be due to the fact that they share five amino acids.

Next, the generated T cell lines were assessed in an in vitro ELISPOTassay to determine whether they could recognize a panel of breast cancercell lines that express varying levels of HER-2/neu at their surface. Inall cases, the p373-382-generated CD8⁺ T cells were able to recognizethe breast cancer cells at much higher levels compared to thep369-377-generated CD8⁺ T cells and the control FLU CD8⁺ T cells (FIG.3B). BT20 cells express HER-2/neu, but do not express HLA-A2 and thusserved as a negative control. These results indicate that breast cancercells express p373-382 on their surface in the context of HLA-A2 andthat CD8⁺ T cells generated using p373-382 have the ability to recognizethese cancer cells.

Another in vitro assay was performed to measure lysis of the breastcancer cells by the T cells. Again, the CD8⁺ T cells generated using thep373-382 polypeptide recognized and lysed all tested breast cancer celllines at much higher levels compared to the CD8⁺ T cells generated usingthe p369-377 polypeptide (FIG. 3C). In this assay, BT20 cells were anegative control as well as the FLO cells, which express HLA-A2, but donot express HER-2/neu.

Lastly, in order to confirm that the p3737-382 polypeptide wasactivating CD8 T cells in an HLA restricted manner, T cell linesgenerated with the three polypeptides used in FIG. 3 were assayed forpeptide-specific reactivity or lytic activity in the presence ofneutralizing HLA-A2 or HLA-ABC monoclonal antibodies. As shown in FIG.4A-B, the reactivity, as assessed by IFN-γ release, of thep373-382-generated T cells was markedly suppressed by inclusion ofeither antibody as compared to T cells treated with control isotypedmatched antibody. In parallel, it also was observed that neutralizingHLA-A2 or HLA-ABC monoclonal antibodies blocked lysis of tumor cells byp373-382-generated T cells as shown in FIGS. 4C-D, respectively.

The findings described in the preceding three paragraphs were repeatedtwo to four times using T cells generated from two to three HLA-A2⁺donors.

Algorithms were used to determine the potential for other HLA alleles,in addition to HLA-A2, to bind to p373-382 or some other embeddedpolypeptide. The algorithms used were SYPEITHI and NetMHCpan. Theresults of this investigation suggest that p373-382 or some fragmentsmay bind other HLA class molecules (Table 2).

TABLE 2 Epitopes predicted within p373-382 HLA Polymorphism OctamersNonamers Decamers HLA-A*0201 — — SLAFLPESFD (SEQ ID NO: 1) HLA-A*03 — —SLAFLPESFD (SEQ ID NO: 1) HLA-A*1101 — — SLAFLPESFD (SEQ ID NO: 1)HLA-A*2402 — SLAFLPESF — (SEQ ID NO: 11) HLA-A*26 — SLAFLPESF — (SEQ IDNO: 11) HLA-B*08 LAFLPESF SLAFLPESF — (SEQ ID NO: 21) (SEQ ID NO: 11)HLA-B*14 — SLAFLPESF — (SEQ ID NO: 11) HLA-B*1501 — SLAFLPESF SLAFLPESFD(SEQ ID NO: 11) (SEQ ID NO: 1) HLA-B*18 SLAFLPES SLAFLPESF — (SEQ ID NO:22) (SEQ ID NO: 11) HLA-B*2705 — SLAFLPESF — (SEQ ID NO: 11) HLA-B*37LAFLPESF SLAFLPESF — (SEQ ID NO: 21) (SEQ ID NO: 11) HLA-B*4402 —SLAFLPESF — (SEQ ID NO: 11) HLA-B*5101 LAFLPESF LAFLPESFD — (SEQ ID NO:21) (SEQ ID NO: 10) HLA-C*01041 LAFLPESF — — (SEQ ID NO: 21) Algorithm:SYFPEITHI [world wide web at “syfpeithi.de/”], Threshold Score: 10Algorithm: NetMHCpan [world wide web at “cbs.dtu.dk/”], Threshold 5% —None

Taken together, the results provided herein demonstrate that p373-382(SLAFLPESFD; SEQ ID NO:1) serves as a prime candidate for cancervaccines and therapeutics for HER-2/neu patients. p373-382 is processedin vitro by cellular enzymes, and it binds a prevalent MHC class Imolecule, HLA-A2. CD8⁺ T cells from human blood can be generated againstthis polypeptide, and these T cells can recognize breast cancer cells,indicating that breast cancer cells are naturally processing p373-382from the expressed HER-2/neu polypeptide and presenting p373-382 on thecell surface in the context of HLA-A*0201.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A composition comprising an immunogenicallyeffective amount of a polypeptide and an adjuvant selected from thegroup consisting of a CpG oligonucleotide, monophosphoryl lipid A, andMontanide ISA-51, wherein the sequence of said polypeptide consists ofthe amino acid sequence set forth in SEQ ID NO:1.
 2. The composition ofclaim 1, wherein said adjuvant is said CpG oligonucleotide.
 3. Thecomposition of claim 1, wherein said adjuvant is said monophosphoryllipid A.
 4. The composition of claim 1, wherein said adjuvant is saidMontanide ISA-51.